Use of microorganisms for the prevention and treatment of intestinal diseases

ABSTRACT

The invention relates to acetylcholine-producing microorganisms for use in the prevention and/or treatment of intestinal diseases, and/or reduction of risks of intestinal diseases, and/or improvement of intestinal health as well as promoting healthy gut flora. The acetylcholine-producing microorganisms may be provided as a pharmaceutical dosage form or as additive to functional food or food supplemental products. Also encompassed is a method for the production of acetylcholine by use of  Lactobacilli . Further the invention refers to microbially produced acetylcholine for use in the treatment and/or prevention of intestinal diseases.

The invention relates to acetylcholine-producing microorganisms for usein the prevention and/or treatment of intestinal diseases, and/orreduction of risks of intestinal diseases, and/or improvement ofintestinal health as well as promoting healthy gut flora. Theacetylcholine-producing microorganisms may be provided as apharmaceutical dosage form or as additive to functional food or foodsupplemental products. Also encompassed is a method for the productionof acetylcholine by use of Lactobacilli. Further the invention refers tomicrobially produced acetylcholine for use in the treatment and/orprevention of intestinal diseases.

A large number of patients suffer from gastrointestinal disordersassociated with the lower small bowel and/or large bowel. Thesedisorders include irritable bowel syndrome (IBS), or spastic colon,idiopathic alterative colitis, mucous colitis, collagenous colitis,Crohn's disease, inflammatory bowel disease in general, microscopiccolitis, antibiotic-associated colitis, idiopathic or simpleconstipation, diverticular disease, and AIDS enteropathy.

Irritable bowel syndrome is the most common of all gastrointestinaldisorders, affecting 11-14% of adults and accounting for more than 50%of all patients with digestive complaints. (G. Triadafilopoulos et al.,Bowel Dysfunction in fibromyalgia, Digestive Dis., Sci. 36 (1): 59-64[1991]; W. G. Thompson, Irritable Bowel Syndrom: Pathogenesis andManagement, Lancet, 341:1569-1572 [1993]). It is thought that only aminority of people with IBS actually seek medical treatment. Patientswith IBS present with disparate symptoms, for example, abdominal pain,predominantly related to defecation, alternating diarrhea andconstipation, abdominal distention, gas, and excessive mucus in thestool. There are three groups of IBS: constipation-predominant IBS(C-IBS), alternating IBS (A-IBS) and diarrhea-predominant IBS (D-IBS).IBS is recognized as a chronic condition, which may have profound effecton the patient's quality of life.

A number of possible causes for IBS have been proposed such asfiber-poor Western diet, intestinal motility malfunction, abdominal painperception, abnormal psychology or behavior, or psycho-physiologicalresponse to stress. However, none of those causes has been fullyaccepted (W. G. Thompson [1993] supra).

Patients suffering from IBS appear to perceive normal intestinalactivity as painful. For example, IBS patients experience pain at lowervolumes of rectal distention than normal or have lower than normalthreshold for perceiving migrating motor complex phase III activity (W.E. Whitehead et al., Tolerance for Rectosigmoid Distention in IrritableBowel Syndrom, Gasteroenterol. 98:1187-92 [1990]; J. E. Kellow et al.,Enhanced Perception of Physiological Intestinal Motility in theIrritable Bowel Syndrom, Gasteroenterol. 101 (6):1621-24 [1991]).

Bowel motility in IBS patients differs from a normal controlled responseto various stimuli such as drugs, hormones, food, and emotional stress(D. G. Wangel and D. J. Deller, Intestinal Motility in Man, III:Mechanisms of Constipation and Diarrhea with Particular Reference to theIrritable Bowel, Gasteroenterol. 48: 69-84 [1965]; R. F. Harvey and A.E. Read, Effect of Cholecystokinin and Colon Motility and on Symptoms ofPatients with Irritable Bowel Syndrome, Lancet i: 1-3 [1973]; R. M.Valori et al., Effects on Different Types of Stress and “Prokineticdrugs” on the Control of the Fasting Motor Complex in Humans,Gasteroenterol. 90: 1890-900 [1986]).

Evans at al. and Govath and Farthing recognized that irritable bowelsyndrome is frequently associated with disordered gastro-intestinalmotility. (P. R. Evans et al., Gastroparesis and Small Bowel Dysmotilityin Irritable Bowel Syndrome, Dig. Dis. Sci. 42 (10): 2087-93[1997]; D.A. Gorard and M. J. Farthing, Intestinal Motor Function in IrritableBowel Syndrome, Dig. Dis. 12 (2): 72-84[1994]). Treatment directed tobowel dysmotility in IBS includes the use of serotonine antagonists (D.P. Becker et al., Mesoazacyclic Aromatic Acid Amides And Esters asSerotonergic Agents, U.S. Pat. No. 5,612,366; M. Ohta at al., Methodsfor Treatment of Intestinal Diseases, U.S. Pat. No. 5,547,961) andCholeocytokinin antagonists (Y. Sato et al., Benzodiazepine derivatives,U.S. Pat. No. 4,970,207; H. Kitajima et al., Thienylazole Compound andThienotriazolodiazepine Compound, U.S. Pat. No. 5,760,032). Colonicmotility index, altered myoelectrical activity in the colon and smallintestinal dysmotility, however, have not proven to be reliablediagnostic tools because they are not IBS-specific (W. G. Thomson[1993],supra).

Administration of probiotics for the treatment of IBS has beenattempted. For example, Allan at al. described the use of a strain ofEnterococcus faecium to alleviate symptoms. (W. D. Allan at al.,Probiotic Containing Enterococcus faecium strain NCIMB 40371 U.S. Pat.No. 5,728,380 and Probiotic, U.S. Pat. No. 5,589,168). Borody taught amethod of treating irritable bowel syndrome by at least partial removalof the intestinal microflora by lavage and replacement with a newbacteria community introduced by fecal inoculum from a disease-screenedhuman donor or by a composition comprising Bacterioids and Escherichiacoli species. (T. J. Borody, Treatment of Gastro-Intestinal Disorderswith a Fecal Composition of Bacterioids and E. coli, U.S. Pat. No.5,443,826).

It is a contention of many scientists that the health and well-being ofpeople can be positively or negatively influenced by the microorganismswhich inhabit the gastrointestinal tract, and in particular the largeintestine. These microorganisms, through the production of toxines,metabolic by-products and short-chain fatty acids, and the like, affectthe physiological condition of the host.

The constitution and quantity of the gut microflora can be influenced byconditions or stress induced by disease, life-style, travel and otherfactors. If microorganisms which positively effect health and well-beingof the individual can be encouraged to populate in the large bowel, thisshould improve the psychological well-being of the host.

The introduction of beneficial microorganisms or probiotics may beaccomplished by ingestion of the organisms in drinks, yogurts, capsules,and other forms allowing viable organisms to arrive at the large bowel.

However, until now, no reliable method has been found or developed tostimulate the enteric nervous system (ENS) which regulates theintestinal bowel movements and secretory functions of epithelial layerin a sufficient manner.

The problem of the present invention was therefore to provide a methodto intervene in the regulation of the intestinal bowel movements andsecretory functions of epithelial layer in a sufficient manner toinfluence thereby the course of intestinal diseases.

The inventors of the present invention have conducted intensive studiesand found as a result that intestinal function can be modulated byacetylcholine-producing microorganisms. Thereby a method for thetargeted dual stimulation of motility and secretion by acetylcholine inthe intestine through selection and specific administration ofacetylcholine-producing microorganism, particularly lactic acid bacteriais provided. This treatment method is a promising alternative oraddition to known therapies for chronic IBS and other disordersassociated with impaired intestinal motility and secretion.

A first aspect of the present invention is therefore anacetylcholine-producing microorganism for the use in the preventionand/or treatment of intestinal diseases and/or reduction of risk ofdeveloping intestinal diseases.

A further aspect of the present invention is the non-medical use of anacetylcholine-producing microorganism for the maintenance and/orimprovement of intestinal health, particularly for the improvement ofintestinal health.

The acetylcholine-producing microorganism is a live organism andpreferably capable of propagating in the intestinal area.

The term “intestinal area” as used herein is intended to include thesmall intestine and large intestine. Large intestine is intended toinclude the colon and rectum, and in humans, is intended to include thecolon, rectum and caecum.

The term “reduction of risk of developing intestinal diseases” as usedherein means that an individual being treated with theacetylcholine-producing microorganism of the present invention exhibitsa lower risk to develop an intestinal disease caused by external stimulior physiological processes compared to a non-treated individual.

The term “maintenance and/or improvement of intestinal health” as usedherein means that an individual, upon treatment with theacetylcholine-producing microorganism, exhibits a different gut flora,which is beneficial for human or animal health and reasonable for amaintenance and/or an improvement of the digestion of said individual.The improved gut flora further may lead to an increased resistance ofthe subject to develop an intestinal disease by out-competing harmfulbacteria and stimulating the normal bowel movement.

The term “microorganism” as used herein comprises bacteria and yeasts.The bacteria are preferably Lactobacillaceae such as Lactobacillusstrains, in particular Lactobacillus sanfranciscensis strains,Lactobacillus rossiae strains, Lactobacillus lactis and Lactobacillusplantarum (Stephenson et al., The production of acetylcholine by astrain of Lactobacillus plantarum,

In certain embodiments, the microorganism is not a Lactobacillusplantarum strain, particularly not Lactobacillus plantarum strain 299v(DSM 9843), or Lactobacillus plantarum strain (ATCC 10241). In certainembodiments, the microorganism is not a Lactobacillus rhamnosus strain,particularly not Lactobacillus rhamnosus GG.

The intestinal diseases encompass inflammatory bowel diseases (IBD),such as ulcerative colitis, Crohn's disease, collagenous colitis,lymphocytic colitis, ischaemic colitis, Behçet's disease, indeterminatecolitis, diversion colitis, pouchitis or microscopic colitis and/orcolon cancer and/or diseases associated with microorganisms, such ascandidiasis, small intestinal bacterial overgrowth, acute or chronicbowel infections, and/or diseases induced by sulphate-reducing bacteria,and/or intestinal diverticular, and/or intestinal carcinoma, and/orfunctional bowel disorders (FBD) such as irritable bowel syndrome,and/or disorders associated with the secretions of the intestinal wallcontrolled by the enteronervous system.

The term “Functional bowel disorder” (FBD) refers to gastro-intestinaldisorders which are chronic or semi-chronic and which are associatedwith bowel pain, disturbed bowel function and social disruption.Particular combinations and prevalence of symptoms characterize infollowing seven FBD subgroups, which are defined in accordance with theclassification system known as the “Rome criteria”: 1) C1:constipation-predominant irritable bowel syndrome; 2) C1:diarrhea-predominant irritable bowel syndrome; 3) C3: Functionalconstipation; 4) C4: Functional diarrhea; 5) C2: Functional abdominalbloating; 6) F3a: Pelvic Floor dyssynergia; 7) F3b: internal analsphincter dysfunction.

More specifically, the intestinal disease may be a functional intestinaldisorder and/or a disorder associated with the secretions of theintestinal wall controlled by the enteric nervous system, in particularfunctional constipation, functional diarrhea and/or irritable bowelsyndrome (IBS), such as particular constipation-predominant IBS,alternating IBS or diarrhea-predominant IBS.

The acetylcholine-producing microorganism of the present invention ispreferably useful for maintaining and/or promoting a healthy gut floraand/or reducing the toxic effects of the digestive process and/orstimulating the digestive system and/or improving intestinal control.The promotion of a healthy gut flora leads to an out-competing of theharmful bacteria in the intestines, in particular the large intestine,and more particularly the colon, and thereby reducing the toxic effectof the digestive process, stimulating the digestive system and improvingbowel control.

The acetylcholine-producing microorganism of the present invention ispreferably useful for modulating the course of inflammatory boweldiseases (IBD) in a beneficial way and relieving the symptoms of IBDpatients by inhibition of the secretion of pro-inflammatory chemokineIP-10.

In another embodiment, the intestinal disease may be an inflammatorybowel disease. The diseases are preferably ulcerative colitis, Crohn'sdisease, collagenous colitis, lymphocytic colitis, ischaemic colitis,Behçet's disease, indeterminate colitis, diversion colitis and/ormicroscopic colitis. The diseases are more preferably ulcerative colitisand/or Crohn's disease.

The microorganism of the present invention is capable of producingacetylcholine. Preferably, the acetylcholine-producing microorganismproduces ≧20, 25, 30, 35 or 40 mg/kg acetylcholine under suitableculture conditions as described, more preferably ≧40 mg/kg and even morepreferably ≧35 mg/kg. Any culture medium might be used for the cultureof the acetylcholine-producing microorganisms, which are suitable forLactobacilli-culture. Preferably, MRS-broth is used as a culture medium.Preferably, the acetylcholine concentration is adjusted to a 10⁶/mlbacteria count.

The acetylcholine-producing microorganism is preferably a bacterium.More preferably, the bacterium is a Lactobacillaceae such as aLactobacillus strain, in particular a Lactobacillus sanfranciscensisstrain, Lactobacillus rossiae strain, Lactobacillus brevis strain, orLactobacillus plantarum strain, which are generally available from thepublic catalogue of Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (Braunschweig, Germany).

In a more preferred embodiment, the Lactobacillus strain is selectedfrom any one of strains DSM 26024, DSM 23090, DSM 23091, DSM 23200, DSM23092, DSM 23093, DSM 23201, DSM 23174 and DSM 23121 or a straincultivated therefrom, more particularly strain DSM 23090 or DSM 23093 ora strain cultivated therefrom. These strains have been deposited at theLeibnitz-Institut DSM—Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (Braunschweig, Germany), according to the BudapestTreaty. The novel Lactobacillus strains have the following accessionnumbers and deposition dates: DSM 23090 (2012-06-21), DSM 23091(2012-06-21), DSM 23200 (2012-06-21), DSM 23092 (2012-06-21), DSM 23093(2012-06-21), DSM 23201 (2012-06-21), DSM 26024 (2012-06-04), DSM 23174(2012-06-21), DSM 23121 (2012-06-21).

The term “strain cultivated therefrom” as used herein refers tooffspring strains derived by cultivation of the original strain.

The acetylcholine-producing microorganism is preferably anacetylcholine-secreting microorganism. The term “acetylcholine-secretingmicroorganism” as used herein means that the microorganism secretsacetylcholine into the culture medium.

The present invention refers to the medical use of anacetylcholine-producing microorganism. Preferably, the microorganism isprovided as a pharmaceutically acceptable dosage form or in form of anutrient, e. g. food or beverage.

In one embodiment, the acetylcholine-producing microorganism is providedin a pharmaceutical composition. The acetylcholine-producingmicroorganisms can also be provided as an additive in a functional foodor in a functional beverage. The pharmaceutical composition, food orbeverages, incorporating the microorganism can be safely consumed andare especially recommended for subjects perceived to be at risk orsuffering from gastro-intestinal dysfunction or organic disorders ordiseases, e.g. conditions or symptoms related to IBS, or IBD. Theycontain the acetylcholine-producing microorganism preferably in aneffective amount to treat or prevent said disorders or diseases.

As used herein, the term “an effective amount” refers to an amounteffective to achieve a desired therapeutic effect, such as treatingand/or preventing diseases, conditions and symptoms related to IBS orIBD.

The effective amount of the acetylcholine-producing microorganismspreferably comprises a dosage in the range of 10⁶ to 10¹² cfu/dosageform (colony forming units/dosage form), more preferably in the range of10⁷ to 0.5×10¹² cfu/dosage form, even more preferably in the range of10⁹ to 10¹¹ cfu/dosage form. The dosage form may be administered once orseveral times, e. g. 2, 3 or more times daily.

The pharmaceutical composition may be in liquid or solid form. Thecomposition contains at least one of the acetylcholine-producingmicroorganisms or a mixture thereof and optionally a pharmaceuticallyacceptable carrier.

The amount of, e. g. microorganisms, incorporated into a pharmaceuticalcomposition may vary from about 0.1 to about 100% by weight, preferablyfrom about 2 to about 20% by weight, even more preferably from about 4to about 10% by weight, based on the total weight of the composition.

The pharmaceutical composition of the present invention can be made inthe usual pharmaceutical forms known in literature, such as for exampletablets, coated tablets, capsules, packets, solutions, suspensions,emulsions, suppositories, pellets, syrups, vaginal suppositories,ointments, creams and so on. Preferably the composition comprises anenteric coating. They can be prepared in the usual manner by mixing theactive ingredient with excipients and/or carriers, optionally addingadjuvants and/or dispersing agents. Should water be used as a diluent,also other organic solvents can be used in the form of adjuvants.Adjuvants can be e. g. water, non-toxic organic solvents such asparaffins, vegetable oils (peanut oil or sesame oil), alcohols (e. g.ethanol, glycerol), glycols (propylene glycol, polyethylene glycol).Solid carriers can be e. g. natural mineral flours (kaolin, talc),synthetic mineral flours (e. g. silicates), sugar (e. g. cane sugar).Emulsifiers can be alkyl sulphonates or aryl sulphonates and the like,dispersers e. g. lignin, methyl cellulose, starch and polyvinylpyrrolidine and lubricants e. g. magnesium is stearate, talc, stearicacid, sodium lauryl sulphonate.

The composition may contain the acetylcholine-producing microorganismslyophilized, pulverized and powdered, optionally for reconstitution in apharmaceutically acceptable liquid carrier administered to intestinalarea, e. g. oral, rectal or naso-duodenal. The administration takesplace in the usual manner, preferably by oral/rectal route. It may thenbe infused, dissolved such as in saline, as an enema. As a powder, itcan preferably be provided in a palatable form for reconstitution fordrinking. The powder may also be reconstituted to be infused vianaso-duodenal infusion.

Pharmaceutical forms adapted to this end can contain, in addition tousual excipients such as lactulose, dextrose, lactose, other additivessuch as sodium citrate, calcium carbonate, calcium dihydrogen phosphate,together with several additional substances such as starch, gelatin andthe like. In case of liquid forms compatible coloring agents orflavoring substances may be added.

Further components of the composition containing theacetylcholine-producing microorganism may include an active agent, e. g.glutamin/glutamate or precursors thereof, mannans, galacturonic acidoligomers, herbal extracts such as Regulat® (registered trademark of DrNiedermayer Pharma) and Iberogast® (registered trademark of SteigerwaldArzneimittelwerk GmbH), chokeberry beer yeast, a drug useful for thetreatment of ulcerative colitis, such as sulphasalazine, 5-ASA agents,corticosteroids, such as adrenal steroid, prednisone, hydrocortisone, orbudesonide, or drugs used against pain, diarrhea, infection or IBS suchas serotonine-4 receptor agonist, e. g. tegaserod. The composition canbe combined with other adjuvants such as antacids to dampen bacterialinactivation in the stomach. Acid secretion in the stomach could also bepharmacologically suppressed using H2 antagonists or omeprazole.

The composition of the invention may be provided in the form of a kitfor is separate sequential or simultaneous administration in conjunctionwith such active agents as described herein above. These active agentsmay conveniently be formulated together with the composition of theinvention in standard pharmaceutical dosage forms, e. g. in combinationwith at least one pharmaceutically acceptable carrier.

In another preferred embodiment, a composition containing theacetylcholine-producing microorganism can contain at least oneadditional microorganism, i.e. non-acetylcholine-producing microorganismsuch as a bacterium for maintaining and/or restoring favorable gutflora. The additional bacteria are preferably pro-biotic bacteria.

In another embodiment of the invention, the acetylcholine-producingmicroorganisms can preferably be provided as a probiotic, preferably asan additive to functional food or to functional beverages.

The amount of the microorganisms as a nutrient additive may vary fromabout 0.0001 to about 20% by weight, preferably from about 0.01 to about10% by weight and even more preferably from about 0.1 to about 5% byweight.

In another preferred embodiment the acetylcholine-producingmicroorganisms can be applied to an edible material, preferably afood—or feed product such as cereals, in particular oat flakes or bread,a beverage or dairy products, in particular yogurt, sauerkraut juice,plant extracts such as Regulat®, fermented beverages or Brottrunk®. Theapplication to the edible material may be achieved by coating saidmaterial with the acetylcholine-producing microorganisms, preferably byspraying the microorganisms onto the edible material. Theacetylcholine-producing microorganisms can also be applied by injectingthem into an edible material, such as bread, yogurt, or cheese,preferably sour dough bread.

The term “functional food” as used herein is food, which, in addition toits nutritional and sensory functions, has a positive effect on themetabolism and, within a balanced nutrition, contributes to animprovement of health, an increase in wellbeing and/or a reduction ofhealth risks. The functional food may be a natural food or a food whichhas been altered by adding or cutting off a component.

In a preferred embodiment, the functional food is a fermented product,more preferably, the functional food is sour dough or sour dough bread.

The sour dough bread of the present invention has the advantage that itmay not only contain the acetylcholine-producing strains, but inaddition also high contents of other acetylated compounds such asN-acetyl-glycine, homoserine, canavanine, and the like.

The term “functional beverage” as used herein is a beverage, which inaddition to its nutritional and sensory function, has a positive effecton the metabolism and, within a balanced nutrition, contributes to animprovement of health, an increase in wellbeing and/or a reduction ofhealth risks. It takes its effect within normal food habits in an amountcommon for consumption.

The functional beverage may comprise additional components such asherbs, vitamins, minerals, aminoacids, or additional other food orvegetable ingredients to provide specific health benefits that go beyondgeneral nutrition. Alternatively, it may include stimulants such astaurin, glucoronolactone, caffeine, B-vitamins, guarana, ginseng, gingkobiloba, L-carnitine, sugars, antioxidants, yerba maté, creatine, milkthistle and the like. In a preferred embodiment, it additionallycontains a prebiotic suitable to be digested by theacetylcholine-producing microorganisms of the present invention.

A preferred functional beverage is a drinking yogurt, a fermented grainbeverage, alcohol-free beer, Brottrunk®, fruitjuice-based beverages orbeverages containing plant or herbal extracts such as Iberogast®.

The functional food or functional beverage can preferably also beadministered with at least one additional active agent for the treatmentof intestinal diseases. The additional active agent can be selected fromthe group of medicaments as described above.

In another embodiment the active agent may preferably be at least oneadditional bacterium for maintaining and/or restoring a favorable gutflora. The additional bacterium can be selected form the group ofprobiotic bacteria.

Preferred pro-biotic bacteria can be selected from the group comprisingStrains from Lactobacillus and Bifidobacterium such as Lactobacillusacidophilus, Lactobacillus johnsonii, Lactobacillus casei, Lactobacilluslactis, Lactobacillus reuteri, Lactobacillus rhamnosus and/orBifidobacterium lactis.

Optionally, the composition can also contain a prebiotic. As usedherein, a “prebiotic composition” is an at least non-digestible foodingredient that beneficially affects the host by selectively stimulatingthe growth, activity or both of one of a limited number of species ofmicroorganisms already resident in the colon. The prebiotic ispreferably a non-digestible oligosaccharide such asfructo-oligosaccharides, galacto-oligosaccharides, lactolose,xylo-oligosaccharides, isomalto-oligosaccharides, soy beanoligosaccharides, gentio-oligosaccharides, gluco-oligosaccharides,fructans, lactosuccrose, short-chain fructo-oligosaccharides, andmixtures thereof.

The present invention also provides a method for the production ofacetylcholine by use of Lactobacilli. Preferably, a single Lactobacillusstrain is used or a combination of Lactobacillus strains or acomposition of Lactobacillus strains. The Lactobacillus strains usedtherefore are in particular Lactobacillus sanfranciscensis strains,Lactobacillus rossiae strains, Lactobacillus brevis strains, orLactobacillus plantarum strains, more particularly strain DSM 26024, DSM23090, DSM 23091, DSM 23200, DSM 23092, DSM 23093, DSM 23201, DSM 23174and DSM 23121 or a strain cultivated therefrom, even more particularlystrains DSM 23090 or DSM 23093.

Another aspect of the present invention is microbially producedacetylcholine for use in the treatment and/or prevention of intestinaldiseases as described above. The acetylcholine may be producedpreferably by the microorganisms of the invention. The microorganismsare preferably Lactobacillaceae such as Lactobacillus strains, inparticular Lactobacillus sanfranciscensis strains, Lactobacillus rossiaestrains, Lactobacillus brevis strains, or Lactobacillus plantarumstrains. The intestinal diseases encompass the intestinal diseasesdescribed above such as inflammatory bowel diseases or functional boweldisorders. Preferably, the disease is irritable bowel syndrome and/ordisorders associated with secretions of the intestinal wall controlledby the enteronervous system. The microbially produced acetylcholine maybe administered orally, e.g. added to food and/or feed products. Theaddition may occur during the manufacture of the food and/or feedproduct by using the microorganisms of the invention also for thefermentation of the food and/or feed product. Such a fermented foodproduct might be sour dough bread, wherein the live microorganisms arekilled during the baking phase by heat, resulting in a sour dough breadcontaining acetylcholine produced by the microorganisms. The content ofthe microbial produced acetylcholine is in a range of about 5 to 1000 mgacetylcholine/kg food or feed product, preferably in a range from about20 to 500 mg acetylcholine/kg feed product or food product, morepreferably in a range from about 40-200 mg acetylcholine/kg feed productor food product. Preferably, the amount of acetylcholine equates to therecommended daily dosage.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Metabolite profile in water extracts of sourdough, sourdoughbread and analog bread. A: Heatmap of absolute metabolite concentrationsin water extract from sourdough (SD), sourdough bread (BR) and analogbread (AN) in triplicates. Water extracts constitute 5-10% of totaldough or bread is dry mass. B: Principal component analysis (PCA) ofmetabolites reveals significant differences in metabolite concentrationsbetween sourdough (SD), sourdough bread (BR) and analog bread (AN).

FIG. 2: Sourdough extracts effectively stimulate stomach muscle motilityby acting directly on muscarinic acetylcholine receptor (mACHR). A:Muscle tone change induced by either acetylcholine (ACH) (2.5 μM) orextracts of sourdough (SD), sourdough bread (BR) or analog bread (AN BR)at 0.02%. Increase in tone was immediately observed upon addition of thestimulants (arrow sign) except for extract of analog bread. B: Median(n>4) change of muscle tone upon differential treatments. Thepro-kinetic effect of acetylcholine (ACH) and extracts of sourdough(SD), sourdough bread (BR) or analog bread (AN BR) is completelyabolished by mACHR specific antagonist atropine. This indicates thatacetylcholine in extracts directly acts on mACHR.

FIG. 3: Tetrodotoxin (TTX) has no significant effect on the stimulationof muscle contraction by sourdough (SD)-derived acetylcholine (ACH). Thefigure shows the muscle tone change stimulated by either acetylcholine(2.5 μM) or 0.02% extracts of sourdough (SD), sourdough bread (BR) oranalog bread (AN BR), and the effect of TTX pre-treatment. TTX did notexhibit significant effect on the muscle contraction induced bysourdough ACH suggesting stimulation is induced by direct action onmuscle mACHR without neuronal mediation.

FIG. 4: Sourdough and sourdough bread extracts stimulate chloride ionssecretion when applied from either serosal or mucosal side of intestinalmucosa. A: Ussing chamber set-up with mucosa piece separating the twochambers. The lower set of electrodes measure transepithelial voltage(V_(TE)) and the lateral set—the short circuit current (I_(SC)). Thesecretion is measured estimated by the change in I_(SC) necessary tomaintain V_(TE) at 0 mV. B: Representative I_(SC) traces stimulated byacetylcholine (ACH), sourdough (SD), sourdough bread (SD BR) or analogbread (AN BR) extracts. Area under the curve is calculated using anintegral (ρA*s/cm²) where blue shows response to extracts andred-striped shows response to electric field stimulation (EFS). Figure Cshows median (n>4) value of change in I_(SC) upon treatment on eithermucosal side (MUC) or serosal side (SER) of guinea pig colonic mucosa.Acetylcholine (ACH) and sourdough (SD) as well as sourdough bread (SDBR) extracts clearly stimulate secretion when applied to either side ofmucosa while analog brad extract has no effect. Showing thatacetylcholine in sourdough and sourdough bread is responsible for thestimulation.

FIG. 5: Atropine completely abrogates chloride ion secretion stimulatedwith sourdough (SD) and sourdough bread (SD BR) extracts. Shown is themean (n>4) value of change in short circuit current (I_(SC)) over timeupon acetylcholine (ACH) (10 μM) and extract (0.1%) application oneither mucosal side (A) or serosal side (B) of guinea pig colonic mucosawith and without atropine pretreatment (1 μM). Atropine completelyabrogated secretion stimulation suggesting the role of mACHR in thesecretory effect of sourdough extracts.

FIG. 6: Atropine completely abrogates secretion stimulation by ACH andextracts of sourdough (SD), sourdough bread (BR) or analog bread (AN BR)when applied serosally but not mucosally. Shown is the mean (n>4) valueof change in short circuit current (I_(SC)) over time subsequent to ACHand extract treatment on the mucosal side with and without atropinepre-treatment (1 μM). Atropine was added on either serosal or mucosalside. Atropine completely abrogated secretion when applied on serosalbut not mucosal side.

FIG. 7: Metabolic profile of sourdough lactic acid bacteria. A: Heatmapof absolute metabolite values in MRS broth after 24 hour lactic acidbacteria inoculation (mean of three experiments). B: PCA plot ofmetabolites indicates sharp distinction between tested sourdoughbacteria and L. paracasei (LC). C: Acetylcholine (ACH) plays thestrongest role in the separation observed in PCA plot since L. paracaseidoes not produce any Acetylcholine.

FIG. 8: Acetylcholine concentration in MRS media upon 24 hours ofincubation with lactic acid bacteria. A: Acetylcholine concentration inMRS broth upon 24 hour inoculation with 0.25×10⁷ of bacteria/mL. B:Acetylcholine (ACH) concentration adjusted to 10⁶/mL bacteria count inMRS. Concentration was determined using LC-MS/MS by comparing the peakarea to the area of solutions with known concentration of acetylcholine.

FIG. 9: Concentrated conditioned media of sourdough lactic acid bacteriasignificantly inhibits the secretion of interferon inducible protein 10(IP-10) by tumor necrosis factor (TNF)-activated intestinal epithelialcells (IEC). The concentration of IP-10 in the culture media of Mode-kcells as measured by ELISA is shown. The cells were incubated for 24hours with concentrated conditioned media (cCM) (black bar) and cCM plus10 ng/mL TNF (grey bar). L. paracasei (L.p.) expresses Lactocepin PrtPthat is capable of efficiently degrading IP-10 and as expected has thehighest inhibitory activity. The cCM of sourdough lactic acid bacteriasignificantly inhibit IP-10 secretion but to a lesser extent. L.sanfranciscensis strains DSM 23174 and DSM 23200 are the most efficientin inhibiting IP-10 secretion next to L. paracasei.

FIG. 10: Formaldehyde-fixed sourdough lactic acid bacteria significantlyinhibit the secretion of interferon inducible protein (IP-10) byTNF-activated intestinal epithelial cells. The concentration of IP-10 inthe culture media of Mode-k cells as measured by ELISA is shown. Thecells where incubated for 24 hours with 20 MOI of fixed lactic acidbacteria (black bar) and 20 MOI of fixed lactic acid bacteria plus 10ng/mL tumor necrosis factor (TNF) (grey bar). Fixed L. paracasei (L.p.)has, similar to fixed L. sanfranciscensis strains DSM 23090 and DSM23092, the highest inhibitory activity on the IP-10 secretion byTNF-activated intestinal epithelial cells (grey bar) as compared toTNF-activated control.

Further, the invention shall be explained in more detail by thefollowing examples.

EXAMPLES 1) Methods and Materials 1.1) Sourdough and Bread

Sourdough (Vollsauer) was prepared by traditional propagation of type Isourdough rye starter containing the Lactobacilli strains DSM 26024, DSM23090, DSM 23091, DSM 23200, DSM 23092, DSM 23093, DSM 23201, DSM 23174and DSM 23121. The composition of sourdough and sourdough bread is: 71%rye flour, 25% wheat flour, 1.8% salt and 2% bread crumbs (pH 4.5,acidity 9-10). The dough was baked at 298° C. for 1.5 hours. Analogbread was identical to sourdough bread with substitution of sourdoughstarter with 2.5% sodium bicarbonate, 0.13% acetic acid and 1.2% lacticacid.

1.2) Metabolite Analysis

Water extracts (<10 kDa) of sourdough, sourdough bread and analog breadas well as MRS growth media of lactic acid bacteria were subjected toLC-MS/MS analysis for metabolite quantification. MRS media was filteredwith 10 kDa Vivaspin 500 filters (Sartorius Stedim biotech, Goettingen,Germany) before analysis.

Samples were measured using:

Dionex Ultra High Performance Liquid Chromatography UltiMate® 3000(Dionex, Idstein, Germany)

-   -   Pump—HPG-3400SD    -   Degasser—SRD-3400    -   Autosampler—WPS—3000TSL    -   Column oven—TCC-3000SD    -   API 4000 QTRAP, Linear Ion Trap Quadrupole Mass Spectrometer (AB        Sciex, Darmstadt, Germany):    -   Ionization type—electrospray ionization (ESI)    -   Instrument control—Analyst software (AbSciex, Darmstadt,        Germany)    -   Stationary phase: TSKgel Amide-80 3 μm (150×2 mm, Tosoh        Bioscience, Stuttgart, Germany)    -   Stationary phase temperature: 40° C.

Mobile phase: eluent A: acetonitrile/5 mM/L ammonium acetate in water(95 + 5) eluent B: 5 mM/L ammonium acetate in water (95 + 5) gradient: 0min 90% A 10% B 5 min 90% A 10% B 10 min 80% A 20% B 15 min 50% A 50% B18 min  0% A 100% B  21 min  0% A 100% B  24 min 90% A 10% B 30 min 90%A 10% B flow: 200 μl/min

The Chromatograms were analysed with Multiquant 2.0 (AB Sciex,Darmstadt, Germany) and concentrations in the samples were calculatedaccording to the spectra of standards.

1.3) Extraction

Sourdough, sourdough bread and analog bread were freeze-dried andgrinded into powder. 100 g of powdered bread or sourdough wassolubilized in 500 mL distilled water and extracted for 3 hours at 50°C. with constant stirring. The suspension was centrifuged at 9000 rpmfor 20 min. The supernatant was collected and kept at 4° C. The pelletwas re-suspended again in 500 mL distilled water and 3 hour extractionrepeated. After centrifugation the pellet was again re-suspended in 500mL distilled water and extracted overnight. Supernatants after threeextraction steps (total volume of approx. 1.5 L) were pooled togetherand step-wise filtered using Vivaflow 200 cassettes of 0.2 μm, 100 kDaand 10 kDa exclusion thresholds (Sartorius, Goettingen, Germany). Thefiltrates of the 100 kDa and 10 kDa exclusion thresholds were freezedried and re-suspended in distilled water to 25% for in vitro assays. 10g of <10 kDa fraction was extracted from 100 g freeze-dried bread andsourdough.

1.4) Endotoxin Measurement and Clean-Up

Endotoxin concentrations measurement in water extracts from sourdough,sourdough bread and analog bread were determined using Limulus AmebocyteLysate (LAL) Chromogenic Endpoint Assay (Hycult biotech, Uden,Netherlands). The assay was performed according to the manufacturer'sinstructions. Endotoxin contamination in water extracts from sourdough,sourdough bread and analog bread was removed using Detoxi-Gel™ EndotoxinRemoving Columns (Thermo Scientific, Rockford, USA), containing a resinwith immobilized polymyxin B to bind and remove pyrogens from solution.The removal of endotoxin was performed according to the manufacturer'sinstructions.

1.5) ELISA

Interferon inducible protein (IP-10) (murine/human) and (murine)concentrations in cell culture supernatants were determined using theappropriate ELISA kits (R&D Europe, Abington, England) according to themanufacturer's instructions. The ELISA was performed using NuncMaxiSorp® flat-bottom 96 well plates (Greiner Bio-One GmbH,Frickenhausen, Germany). Briefly 96-well plates were coated with theappropriate capture antibody overnight at RT. Plates were washed 3 timeswith phosphate buffered saline (PBS), blocked with 1% bovine serumalbumin in PBS and incubated with cell culture supernatants for 1.5 h atRT. Plates were washed and incubated with the appropriate detectionantibody for 1.5 h at RT. Plates were washed and incubated with adetection enzyme. Plates were washed and incubated with a substratesolution. Protein concentration was determined by photometrical analysisof the reaction of substrate and detection enzyme.

1.6) Bacterial Culture

L. sanfranciscensis strains (DSM 23090, DSM 23091, DSM 23092, DSM 23093,DSM 23174, DSM 23200, DSM 23201) and L. rossiae (DSM 26024) isolatedfrom sourdough, L. sanfranciscensis type strain DSM 20451 (DSMZ GmbH,Braunschweig, Germany), L. plantarum FUA 3038 and L. brevis 3113(provided by Prof. Ganzle from University of Alberta, Canada), L.paracasei VSL#3 (provided by Dr. DeSimone, L'Aquila, Italy) were grownat 30° C. in MRS broth (pH 5.4) containing freshly added 0.15% L-cysteinunder anaerobic conditions using Anaerogen packages (Anaerogen,Basingstoke, Oxoid, UK). Fixed bacteria (5% formaldehyde, 4 hours, 4°C.) were washed three times with sterile PBS before use. Concentratedconditioned media (CM) were generated by transferring bacteria (5×10⁷cfu/ml) from anovernight culture to DMEM (1% glutamine, 20 mM HEPES) andanerobical cultivation overnight at 30° C. Bacteria and bacterialsupernatant (CM) were separated after centrifugation (4500 g, 10 min,RT). CM was adjusted to pH 7.4, filter sterilized (0.22 μm), andconcentrated (100×) using Vivacell filter systems with an exclusion sizeof 100 kDa (Satorius Stedim Biotech, Goettingen, Germany). Concentratedconditioned media was diluted to 1× in the cell culture stimulationexperiments. Agar plates were obtained by adding 1.5% of agar to theabove described respective medium.

1.7) Motility

Motility measurements were performed with corpus circular musclepreparations from Dunkin Hardley guinea pigs (Sulzfeld and HarlanWinkelmann GmbH, Borchen, Germany). Contractile force of the muscle wasmeasured using force transducer in organ bath using LabChart 5 software(ADInstruments, Spechbach, Germany). Briefly, stomach muscle tissue wasdissected from mucosa layer in continuously perfused ice-coldpreparation Krebs solution (pH 7.4) (MgCl₂×6H₂O 1.2 mM, CaCl₂×2H₂O 2.5mM, NaH₂PO₄ 1.2 mM, NaCl 117 mM, NaHCO₃ 25 mM, C₆H₁₂O₆ 11 mM, KCl 4.7mM). A 1.5 cm² piece of corpus circular muscle was cut out and mountedfrom both ends with polyamide thread between two electrodes into organbath in 20 mL experimental Krebs solution (identical to preparationKrebs except for NaHCO₃ 20 mM) at 37° C. and aerated continuously withCarbogen (95% O₂ and 5% CO₂). After an equilibration period of 45 minmuscle is preparations were stimulated by electrical field stimulation(EFS) to test vitality. The change in contractile force during EFS aswell experimental treatment was measured by the force transducer. Thetime lapse between any treatments was always 20 min.

1.8) Ussing Chamber

The ion movement across intestinal epithelia was measured with Ussingchamber technique (Easy mount chambers, Physiologic instruments, SanDiego, USA) and LabChart 5 software (ADInstruments, Spechbach, Germany).Briefly segments, of the distal colon of Dunkin Hardley guinea pigs(Sulzfeld and Harlan Winkelmann GmbH, Borchen, Germany) were dissected,the muscle layers removed and mucosa/submucosa preparations were mountedinto slider with a recording area 0.5 cm². Apical and basolateral sideswere bathed separately in 5 mL Krebs solution. During experimentalprocedures, the bath was maintained at 37° C. and aerated continuouslywith Carbogen (95% 0₂ and 5% CO₂). After an equilibration period of 45min tissue was electrically stimulated (Parameters: stimulus strength6V, duration 10 sec, frequency 10 Hz, single pulse duration 0.5 ms) toassess tissue vitality. For assessment of active ion transportspontaneous occurring transepithelial voltage (V_(TE)) formed by passiveion transport across the tissue was set to 0 mV by applying shortcircuit current (I_(SC)). When the active chloride ion secretion isinduced an increase in I_(SC) is observed necessary to keep V_(TE) at 0mV. The change in I_(SC) is equivalent to the current generated by theanions secretion or cation absorption. Transepithelial resistance(TER=V_(TE)/I_(SC)×1000/2) of tissue was measured at the beginning andat the end of each experiment to assess the tissue integrity.

1.9) Statistical Analysis

Data are expressed as mean values±standard deviation (SD). Allstatistical computations were performed using Statistical programmingplatform R comparing treatment vs. corresponding control group wereanalyzed using unpaired t-tests. Data comparing several treatments vs.corresponding control group were analyzed using One-Way ANOVA followedby an appropriate multiple comparison procedure. If data was notnormally distributed or comprised discontinuous data, non-parametricaltests (Mann-Whitney/Rank sum test, ANOVA on ranks) were used.Differences were considered significant if p-values were <0.05 (*) or<0.01 (**). Principal component analysis (PCA) is described in Pearson,K.; On Lines and Planes of Closest Fit to Systems of Points in Space,Philosophical Magazine (1901), 2 (11), 559-572 and Theodoridis, G.,Gika, H. G., Wilson, I. D.; LC-MS-based methodology for globalmetabolite profiling in metabonomics/metabolomics, TrAC Trends inAnalytical Chemistry (2008), 27 (3), 251-260.

2.) Results

2.1) LC-MS/MS Analysis of Metabolites in Extracts from Sourdough,Sourdough Bread and Analog Bread

To compare the effect of fermentation on sourdough and sourdough breadwater soluble extracts (<10 kDa, triplicates) of raw sourdough,sourdough bread and analog bread prepared from three different batcheswere subjected to LC-MS/MS analysis. The concentration of metabolites inextracts was determined by comparison to the standard solution withknown concentration of metabolites.

Principal component analysis (PCA) demonstrated significant differencesin metabolites isolated from sourdough, sourdough bread and analog bread(FIG. 1). Raw sourdough has significantly higher amounts of free aminoacids reflecting proteolitic acitivity of endogenous flour enzymes aswell as lactic acid bacteria proteases. Before baking fresh unfermentedflour is added to the sourdough explaining why there are no differencesin the free amino acid content between analog and sourdough bread.Acetylcholine is a metabolite that is consistently present in sourdoughand sourdough bread but not in analog bread (Table 1). Fermentation bysourdough bacteria is the only difference between sourdough bread andanalog bread, suggesting acetylcholine is produced by microorganismspresent in sourdough.

TABLE 1 Sourdough and sourdough bread contain high amounts ofacetylcholine of with 38.6 mg/kg in dry bread. Concentration wasdetermined using LC-MS/MS by comparing the peak area to the area ofsolutions with standard concentrations of acetylcholine. ACHconcentration ACH concentration in bread Water extracts in waterextracts or sourdough, dry mass Sourdough, 10 kDa 1819 ± 717 μM 26.5 ±10.4 mg/kg Sourdough bread, 10 2644 ± 273 μM 38.6 ± 4.03 mg/kg kDaAnalog bread, 10 kDa 44.5 ± 1.0 μM 0.64 ± 0.03 mg/kg

2.2) Sourdough-Derived Acetylcholine Triggers Muscle Contraction InVitro

Acetylcholine (ACH) is a neurotransmitter that is responsible for theactivation of motility in the gastro-intestinal tract by stimulatingeither muscarinic (mACHR) or nicotinic ACH receptors (nACHR) on themuscle cells. To determine if the sourdough-derived acetylcholinemimicks this activity, isolated corpus muscle of guinea pig wasstimulated with extracts of sourdough, sourdough bread and analog breadand the contraction stimulation was measured. Both sourdough andsourdough extracts but not analog bread extract induced musclecontractions similar to acetylcholine at equivalent concentration (FIG.2). Atropine, mACHR-specific antagonist, was used to determine whetherextracts stimulate contraction by activating muscarinic or nicotinicACHR. Pre-treatment of muscle strips with atropine completely abrogatedstimulation by ACH as well as by sourdough and sourdough bread extractsindicating that sourdough-derived acetylcholine is acting via mACHR.

Furthermore to clarify whether sourdough-derived ACH acts through theactivation of neurons that consequently stimulate muscle cells, musclepreparations were pre-treated with tetrodotoxin (TTX). TTX blocks actionpotential generated by neurons that abrogates downstream signalling. TTXpre-treatment had no significant effect on muscle contraction induced byacetylcholine and extracts suggesting that both directly activate mACHRon muscle cells (FIG. 3). Motility is one of the crucial functions ofthe GI tract. Agonists and antagonists of serotonin(5-hydroxytryptamine) receptors are a common treatment option formodulating motility and consequently bowel movements of IBS patients(Camilleri, M. and V. Andresen, Current and novel therapeutic optionsfor irritable bowel syndrome management. Dig Liver Dis, 2009. 41(12): p.854-62). These observations show that external application of ACH atlocal sites could also be utilised to modulate ENS-mediated motility.

2.3) Sourdough-Derived Acetylcholine Stimulates Secretion by theIntestinal Mucosa from the Luminal Side

Acetylcholine (ACH), released by the enteric neurons to the serosal sideof the intestinal wall, stimulates secretion of chloride ions by themucosa subsequently driving the passive transport of water into thelumen. This effect is transient due to rapid degradation ofacetylcholine by acetylcholine esterase. The effect of sourdough-derivedACH on the intestinal secretory function was tested in guinea pig colon.Extracts of sourdough, sourdough bread, analog bread as well as (pure)ACH were applied on either luminal (mucosal) or serosal side ofintestinal mucosa/submucosa preparations from guinea pig distal colon.The experiment was performed in Ussing chamber and the change inshort-circuit current (I_(SC)) was measured. In this system, passiveflow of ions across a tissue or epithelial cell layer is eliminated bybalancing electrical, osmotic, hydrostatic and chemical gradients acrossthe preparation, such that only active ion transport is measured. In theUssing chamber, electrodes are placed close to each side of the tissueto allow detection of the spontaneous potential difference (PD) acrossthe epithelium, generated as a consequence of active ion transport(Hirota, C. L. and McKay D. M., Cholinergic regulation of epithelial iontransport in the mammalian intestine, Br. J. Pharmacol., 2006, 149(5):p.463-79). Surprisingly an increase in I_(SC) could be observed when thepreparations were stimulated from both mucosal and serosal side by ACHas well as ACH-containing extracts (analog bread extract had no effect)(FIG. 4), showing that acetylcholine in sourdough and sourdough bread issuitable for the stimulation.

The tissue was pre-treated with atropine and the effect of extracts onsecretion measured again (FIG. 5). Atropine completely abolished theresponse corroborating the role of mACHR in the pro-secretory effect ofsourdough extracts.

Earlier research provides evidence of the expression of ACHR inintestinal epithelial cells on the basolateral side but not on theapical side of the cell layer (Hirota, C. L. and McKay D. M.,Cholinergic regulation of epithelial ion transport in the mammalianintestine, Br. J. Pharmacol., 2006, 149(5):p. 463-79). Therefore it ishighly probable that ACH applied from apical side crosses the cell layerand stimulates the receptors on the basolateral side. This hypothesiswas verified by the fact that when ACH is applied apically, i.e. on themucosal or luminal side, the secretory effect was significantlyinhibited when atropine was applied to the serosal but not to themucosal side (FIG. 6). Atropine applied on the serosal side blocked allmACHR on basolateral side abrogating ACH activity. However, whenatropine is applied on the mucosal side this results into smalleramounts reaching basolateral side and thus only partially inhibiting ACHactivity. The observation that atropine can cross epithelial layer andblock basolateral mACHR was confirmed by the fact that when ACH wasapplied serosally and atropine mucosally secretion was inhibited (datanot shown).

These observations show that external application of ACH at local sitescould also be utilised to modulate ENS-mediated fluid secretion. This isespecially important, since fluid secretion into the intestine ishypothesized to provide the ideal environment for enzymatic digestionand to facilitate the passage of stool through the intestinal tract.Furthermore recent studies suggest that acute and locally targeted watersecretion serves as a protective measure against epithelial damage atpoints of particular mechanical stress (Barrett, K. E. and S. J. Keely,Chloride secretion by the intestinal epithelium: molecular basis andregulatory aspects. Annu Rev Physiol, 2000. 62: p. 535-72 and Sidhu, M.and H. J. Cooke, Role for 5-HT and ACh in submucosal reflexes mediatingcolonic secretion. Am J Physiol, 1995. 269(3 Pt 1): p. G346-51).

2.4) LC-MS/MS Analysis Revealed Presence of Acetylcholine in GrowthMedia of Sourdough Lactic Acid Bacteria

Seven strains of L. sanfranciscensis (DSM 23090-DSM 23201) and L.rossiae (DSM 26024) isolated from sourdough, L. plantarum FUA 3038 andL. brevis 3113 isolated from another sourdoughs, and L. paracasei(VSL#3) were grown for 24 hours in MRS media. The growth media wascollected, filtered and analyzed using LC-MS/MS.

PCA analysis shows significant difference in the metabolites profiles ofall sourdough isolated bacteria compared to L. paracasei. The separationis due in major part to the impact of acetylcholine present in sourdoughbacteria growth media but not in L. paracasei media (FIG. 7). Thehighest ACH producer over 24 hours is L. brevis 3113, which additionallyhas the highest growth rate (FIG. 8A). However when the concentration isadjusted to a bacterial number, e.g. 10⁶/ml, in the broth, the most ACHper bacterial cell is produced by L. sanfranciscensis strains DSM 23090and DSM 23093 (FIG. 8B).

2.5) Sourdough Lactic Acid Bacteria Inhibit Secretion of Chemokine IP-10by TNF-Activated Intestinal Epithelial Cells

Lactocepin PrtP, a serine protease expressed in L. paracasei (VSL#3),selectively degrades pro-inflammatory chemokine interferone inducibleprotein 10 (IP-10). To investigate if PrtP is also present in theLactobacilli of the present invention, the total bacterial DNA wasisolated from eight sourdough strains: L. sanfranciscensis (DSM 23090,DSM 23091, DSM 23092, DSM 23093, DSM 23174, DSM 23200, DSM 23201) and L.rossiae (DSM 26024) as well as L. paracasei as positive control. DNA wasamplified using Lactocepin PrtP specific primers and visualized onagarose gel. No detectable amounts of lactocepin PrtP gene were presentin sourdough-isolated lactic acid bacteria. The eight sourdough lacticacid bacteria strains and L. paracasei were tested for their effects onthe secretion of pro-inflammatory chemokine IP-10 by unstimulated andTNF-activated Mode-K cells. Interestingly, both conditioned media (FIG.9) and fixed lactic acid bacteria (FIG. 10) demonstrated IP-10inhibitory activity despite the fact that no Lactocepin PrtP gene wasdetected. This suggests that there are both secreted and cellsurface-bound factors produced by sourdough lactic acid bacteriasuitable to inhibit secretion of pro-inflammatory chemokine IP-10. Thisresult provide a basis for potential treatment of IBD and relief oftheir symptoms, since IP-10 has been implicated in re-enforcingintestinal inflammation in IBD patients.

1. An acetylcholine-producing microorganism for medical use in theprevention and/or treatment of intestinal diseases, and/or reduction ofrisk of intestinal diseases.
 2. The acetylcholine-producingmicroorganism for use according to claim 1, for maintaining and/orpromoting a healthy gut flora and/or reducing the toxic effects of thedigestive process and/or stimulating the digestive system and/orimproving intestinal control.
 3. The acetylcholine-producingmicroorganism for use according to claim 1, wherein the intestinaldisease is a functional intestinal disorder and/or a disorder associatedwith the secretions of the intestinal wall controlled by the entericnervous system, in particular functional constipation, functionaldiarrhea and/or irritable bowel syndrome (IBS), more particularconstipation predominant IBS, alternating IBS or diarrhea predominantIBS.
 4. The acetylcholine-producing microorganism for use according toclaim 1, wherein the intestinal disease is an inflammatory boweldisease, in particular ulcerative colitis and/or Crohn's disease.
 5. Theacetylcholine-producing microorganism for use according to claim 1,wherein the microorganism produces ≧30 mg/kg acetylcholine undersuitable culture conditions.
 6. The acetylcholine-producingmicroorganism for use according to claim 1, wherein the microorganism isa bacterium.
 7. The acetylcholine-producing microorganism for useaccording to claim 1, wherein the microorganism is a Lactobacillusstrain, in particular a Lactobacillus sanfranciscensis strain, aLactobacillus rossiae strain, a Lactobacillus brevis strain or aLactobacillus plantarum strain, more particularly strains DSM 23090 orDSM
 23093. 8. The acetylcholine-producing microorganism for useaccording to claim 1, wherein the Lactobacillus is selected from any oneof strains DSM 26024, DSM 23090, DSM 23091, DSM 23200, DSM 23092, DSM23093, DSM 23201, DSM 23174 and DSM 23121 or a strain cultivatedtherefrom.
 9. The acetylcholine-producing microorganism for useaccording to claim 1 in a pharmaceutical dosage form, in a functionalfood or in a functional beverage.
 10. The acteylcholine-producingmicroorganism for use according to claim 9, wherein the functional foodis sourdough bread.
 11. The acetylcholine-producing microorganism foruse according to claim 9, further comprising at least one additionalbacterium for maintaining and/or restoring a favorable gut flora. 12.The acetylcholine-producing microorganism for use according to claim 9,further comprising at least one additional medicament for the treatmentof intestinal diseases.
 13. A method for the production of acetylcholineby use of lactobacilli, particularly by the use of lactobacilli selectedfrom Lactobacillus sanfranciscensis and Lactobacillus rossiae strains.14. A method for the treatment and/or prevention of intestinal diseases,in a patient in need of such, comprising orally administeringmicrobially produced acetylcholine to said patient.
 15. Non-medical useof an acetylcholine-producing microorganism for maintenance and/orimprovement of intestinal health.
 16. (canceled)